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Effect of ghrelin on regulation of splenic sympathetic nerve discharge
AUTNEU-01871; No of Pages 4 Autonomic Neuroscience: Basic and Clinical xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Short Communication
Effect of ghrelin on regulation of splenic sympathetic nerve discharge Sivasai Balivada a,b,1, Hitesh N. Pawar a,b,⁎,1, Shawnee Montgomery a, Michael.J. Kenney a,b a b
Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, United States Department of Biological Sciences, College of Science, University of Texas at El Paso, El Paso, TX 79968, United States
a r t i c l e
i n f o
Article history: Received 2 May 2016 Received in revised form 22 July 2016 Accepted 18 August 2016 Available online xxxx Keywords: Ghrelin Splenic SND Lumbar SND Intracerebroventricular injection
a b s t r a c t Ghrelin influences immune system function and modulates the sympathetic nervous system; however, the contribution of ghrelin to neural-immune interactions is not well-established because the effect of ghrelin on splenic sympathetic nerve discharge (SND) is not known. This study tested the hypothesis that central ghrelin administration would inhibit splenic SND in anesthetized rats. Rats received intracerebroventricular (ICV) injections of ghrelin (1 nmol/kg) or aCSF. Lumbar SND recordings provided a non-visceral nerve control. The ICV ghrelin administration significantly increased splenic and lumbar SND, whereas mean arterial pressure (MAP) was not altered. These findings provide fundamental information regarding the nature of sympathetic-immune interactions. © 2016 Published by Elsevier B.V.
1. Introduction Ghrelin is a peptide hormone that was originally isolated from the gastrointestinal system and identified as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a) (Baatar et al., 2011). Subsequent investigations have demonstrated that this peptide can influence a number of physiological systems and responses, including the sympathetic nervous system (SNS) (Lambert et al., 2011; Matsumura et al., 2002). Ghrelin receptors are expressed in the central nervous system, especially in brainstem and hypothalamic sites (Wu et al., 2009), and central ghrelin administration in experimental animals modulates sympathetic nerve discharge (SND) regulation. The intracerebroventricular (ICV) administration of ghrelin has been reported to decrease brown adipose tissue SND in rats (Yasuda et al., 2003), reduce renal SND in rabbits (Matsumura et al., 2002), and produce little effect on renal SND in rabbit offspring from mothers who consumed a normal fat diet compared with enhanced ghrelin-induced renal SND responses in rabbit offspring from mothers fed a high fat diet (Prior et al., 2014). Microinjection of ghrelin in the nucleus of the solitary tract reduces renal SND in rats (Lin et al., 2004). In human subjects direct recordings of efferent muscle sympathetic nerve outflow using microneurography have provided information regarding the effect of peripherally-administered ghrelin on SND regulation. Lambert and colleagues reported that intravenous infusion of ghrelin increased muscle
⁎ Corresponding author at: Department of Biological Sciences, College of Science, University of Texas at El Paso, Biosciences Research Building, Room No. 4.216, El Paso, TX 79968, United States. E-mail address: [email protected] (H.N. Pawar). 1 These authors contributed equally to this work.
SND and reduced arterial blood pressure (Lambert et al., 2011). These investigators speculated that combined baroreflex-mediated and central neural effects of ghrelin may play a role in mediating the observed sympathoexcitatory response. Krapalis et al., observed a biphasic muscle SND response to intravenous ghrelin; an initial sympathoexcitation associated with reduced arterial blood pressure, followed by a progressive decline of muscle SND towards control levels, despite a sustained reduction in arterial blood pressure (Krapalis et al., 2012). These findings indicate that peripheral ghrelin administration can modulate the level of muscle SND in human subjects, although the role of central neural ghrelin in mediating these responses remains unclear. A role for ghrelin in modulating immune system function is wellestablished, primarily as an anti-inflammatory agent and an immunoregulatory hormone (Baatar et al., 2011). Numerous bidirectional pathways provide the foundation for communication between the nervous system and the immune system, and the efferent arm of the SNS plays an important role in mediating neural-immune interactions (Kenney and Ganta, 2014). Physiological activation of splenic SND enhances the expression of splenic cytokine and chemokine genes, an effect that is abrogated by splenic nerve denervation (Ganta et al., 2004), indicating that changes in the level of efferent splenic nerve outflow can influence immune function in a peripheral lymphoid organ. It is intriguing to postulate that ghrelin may play a role in neuroimmune interactions by influencing the SNS, however, the effect of ghrelin on the level of splenic SND is not known. Given the role of ghrelin as an anti-inflammatory molecule, and the influence of splenic SND activation to influence splenic cytokine gene expression, the first goal of the present investigation was to test the hypothesis that ICV administration of ghrelin would inhibit splenic SND. Important with respect to the role of ghrelin in modulating muscle SND in human
http://dx.doi.org/10.1016/j.autneu.2016.08.011 1566-0702/© 2016 Published by Elsevier B.V.
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
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S. Balivada et al. / Autonomic Neuroscience: Basic and Clinical xxx (2016) xxx–xxx
subjects (Krapalis et al., 2012; Lambert et al., 2011), to date there is no information available concerning the effect of central ghrelin on the level of lumbar SND, despite the fact that this nerve innervates the rat hindlimb and tail (Baron et al., 1988). In this regard, lumbar SND recordings may provide translational significance to previous studies that tested the effect of ghrelin on muscle SND in human subjects (Krapalis et al., 2012; Lambert et al., 2011). Therefore, the second goal was to determine the effect of ICV administration of ghrelin on the level of lumbar SND. 2. Methods The experimental procedures and protocols were completed in accordance with the American Physiological Society's guiding principles for research involving animals and approved by the Institutional Animal Care and Use Committee at Kansas State University and at the University of Texas at El Paso. 2.1. General procedures Experiments were completed using male Sprague-Dawley rats (419 ± 15 g). Anesthesia was induced by isoflurane (3–5%) and maintained during surgical procedures using isoflurane (1.25%–1.75%), αchloralose (80 mg/kg, iv), and urethane (400 mg/kg, ip) (Kenney et al., 2013). Isoflurane anesthesia was discontinued following completion of the surgical procedures. During experimental protocols, maintenance doses of α-chloralose (35–45 mg/kg/h) were administered intravenously and maintenance doses of urethane (200 mg/kg, every 4 h) were administered intraperitoneally. The adequacy of anesthesia during the initial surgical procedures was indicated by the absence of a withdrawal reflex in response to mechanical stimulation of the tail or hind limb. Femoral arterial pressure was monitored using a pressure transducer connected to a blood pressure analyzer. Colonic temperature was maintained at 37.5–37.8 °C during surgical interventions by a temperature-controlled table. 2.2. Sympathetic nerve recordings Activity was recorded biphasically with a platinum bipolar electrode after preamplification (bandpass filter, 30–3000 Hz) from splenic and lumbar sympathetic nerves. Sympathetic nerves were identified using a lateral approach and dissected free of surrounding connective tissue, and nerve-electrode preparations were covered with silicone gel to prevent exposure to room air. Sympathetic nerve potentials were full-wave rectified, integrated (time constant 10 ms) and quantified as μvolts × seconds (μV·s) (Hosking et al., 2009). SND recordings were corrected for background noise after administration of the ganglionic blocker chlorisondamine (5 mg/kg, iv) or nerve crush. 2.3. ICV injection Anesthetized rats were placed in a stereotaxic frame. The head was leveled between bregma and lambda, a small hole was made in the skull, and the dura was removed to increase precision of the depth of the injector needle. A Hamilton syringe was loaded onto a Quintessential Stereotaxic Injector (Stoelting Co., Wood Dale, IL), and the needle guided to 1.4 mm lateral from midline, 0.9 mm posterior to bregma, and 3.5 mm below dura. The body weight adjusted ghrelin dose was selected based on previous studies that analyzed the effect of ICV ghrelin injection on SND in animal models (Matsumura et al., 2002; Yasuda et al., 2003). Ghrelin (1 nmol/kg; Phoenix Pharmaceuticals) suspended in artificial cerebrospinal fluid (aCSF) or aCSF alone was administered (10 μL) over 5 min. Fluorescent latex microspheres (50 nm diameter, Lumafluor) were administered immediately before euthanasia to histologically verify that injections were completed in the lateral ventricle.
2.4. Experimental protocols After completion of surgical procedures, anesthetized rats were allowed to stabilize for 60 min before initiation of experimental protocols. Splenic SND, lumbar SND, mean arterial pressure (MAP), and heart rate (HR) were recorded continuously throughout experimental protocols. Following stabilization, pre-injection values were averaged over a 15 min control period (time 0). At the end of the control period rats were administered ghrelin (1 nmol/kg) or aCSF via infusions in the lateral ventricle. Infusions were completed over a 5 min period followed by a 45 min post-infusion period. At the end of experiments rats were euthanized by an intravenous overdose of methohexital sodium (Brevital®) (150 mg/kg, iv). 2.5. Data collection and statistical analysis A computer-based ADInstruments Powerlab data acquisition system was used to collect all experimental data. Values are reported as means ± SE. Splenic and lumbar SND data are expressed as percentage change from control values. MAP and HR are reported as the absolute change from control values. Statistical analyses included two-factor (treatment and time) repeated measures ANOVA with one factor repetition (time). Data that demonstrated statistically significant main and interaction effects were further analyzed with Bonferroni t-tests. The overall level of statistical significance was p b 0.05. 3. Results The ICV injection sites were histologically confirmed to be in the lateral ventricle in all experiments included in the present study. Fig. 1 shows summarized splenic SND (A) and lumbar SND (B) data recorded before infusion (time 0), at the completion of the ICV infusion period (5 min), and for 45 min post ICV administration of aCSF (n = 12) or ghrelin (n = 8). Data are presented as change from control levels at 5 min intervals. Splenic and lumbar SND were progressively and significantly increased from control levels after central ghrelin administration (p b 0.05), with the magnitude of the sympathoexcitation more robust in lumbar SND. In contrast, splenic and lumbar SND remained unchanged from control levels for the duration of the post-infusion period following ICV aCSF administration. Fig. 2 shows summarized MAP (A) and HR (B) data recorded before (time 0), at the completion of the ICV infusion period (5 min), and for 45 min after ICV administration of aCSF (n = 13) or ghrelin (n = 12). MAP did not differ from control levels in either aCSF- or ghrelintreated rats, or between groups, during the 45 min post infusion period (Fig. 2A). Similarly, HR did not differ from control levels following aCSF and ghrelin infusions, although in ghrelin-treated rats HR tended to increase whereas in aCSF-treated rats HR tended to decrease. The change in HR between the two treatments became significant at 10 min postinfusion and continued until the end of the post infusion period (p b 0.05) (Fig. 2B). 4. Discussion The present results indicate that ICV ghrelin administration in anesthetized rats produces a progressive excitation of splenic sympathetic nerve outflow, a response pattern paralleled by a robust ghrelininduced lumbar sympathoexcitation. Central ghrelin administration was not associated with significant changes in arterial blood pressure, suggesting that the sympathoexcitatory responses to ICV ghrelin were not secondary to unloading of the arterial baroreceptors. As expected, each of the measured variables remained unchanged from control values in response to the ICV administration of aCSF. These findings support the notion that, under the conditions of the present experiments, administration of ghrelin into the lateral ventricle modulates the central
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
S. Balivada et al. / Autonomic Neuroscience: Basic and Clinical xxx (2016) xxx–xxx aCSF Ghrelin
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Fig. 1. Changes in (A) splenic sympathetic nerve discharge (SND, %Δ) and (B) lumbar SND (%Δ) responses to the ICV administration of aCSF (n = 12) or ghrelin (1 nmol/kg, n = 8). Time 0 signifies control. ICV ghrelin and aCSF infusions were completed over a 5 min infusion period (shaded in grey). Measurements were maintained for 45 min after administration of ghrelin or aCSF. *Significantly different from control values. †Ghrelin significantly different from aCSF. Values are means ± SE.
hypothesis, in the present study ICV ghrelin administration was associated with splenic sympathoexcitation. This finding does not diminish the fact that ghrelin exerts substantial anti-inflammatory physiological responses, which are mediated by a variety of mechanisms including local and systemic effects; rather it indicates that a central ghrelinmediated splenic sympathoinhibition is likely not a key contributor. Based on the ghrelin-induced activation of lumbar and splenic SND, it might have been expected that arterial blood pressure would be increased in response to ICV ghrelin administration. However, this was not the case and one explanation is that central ghrelin administration may produce nonuniform changes in SND, that is; inhibition of renal SND (Matsumura et al., 2002) and activation of lumbar SND and splenic SND (Fig. 1). Directionally opposite changes in the level of activity in nerves innervating vascular beds that receive substantial levels of cardiac output would be expected to moderate changes in mean arterial blood pressure. However, this is not a complete explanation based on the results of other studies. For example, the reduction in renal SND to central ghrelin administration reported by Matsumura et al. (2002) was associated with a concomitant, marked reduction in arterial blood pressure. On the other hand, Freeman et al. (2013) reported that chronic ICV ghrelin infusion in conscious rats caused a modest reduction in arterial blood pressure (SND was not recorded in this study, and ICV ghrelin administration in rabbit offspring from mothers who consumed a normal fat diet produced modest changes in both renal SND and arterial blood pressure (Prior et al., 2014). At the present time, it is difficult to reconcile differences in arterial blood pressure responses to central ghrelin administration between individual studies, although multiple experimental variances, including; species, anesthesia, ghrelin dose, and ghrelin infusion rates, likely contribute.
regulation of SND by increasing the level of activity in sympathetic nerves innervating two distinct targets. The present study is the first to determine the effects of ghrelin on splenic and lumbar SND, thereby extending the current level of knowledge regarding the role of ghrelin as a modulator of sympathetic neural outflow. The sympathetic innervation to the spleen, via the splenic nerve, provides a neural communication pathway from central nervous system to immunocompetent cells in this lymphoid organ (Felten et al., 1985; Felten et al., 1987). Ghrelin-induced activation of lumbar SND provides information regarding the central effect of this peptide hormone on the sympathetic innervation to the rat hind limb, which may be of translational importance as ghrelin administration modulates muscle SND in human subjects (Krapalis et al., 2012; Lambert et al., 2011). The SNS and the immune system are prominent adaptive physiological systems that for many years were considered to function independently of each other. However, recent lines of inquiry have established a role for the SNS in mediating neural-immune interactions (Kenney and Ganta, 2014). Moreover, it is well-established that ghrelin can exert potent anti-inflammatory effects both in vitro and in vivo, with a promising therapeutic outlook in the treatment of inflammatory diseases (Vanessa et al., 2013). A strategic aspect associated with identifying integrative immune-sympathetic physiological interactions is focused on determining if immune system-related molecules engage supraspinal pre-sympathetic motor neurons. Based on the role of ghrelin as an anti-inflammatory molecule, and the capacity of splenic SND activation to mediate an upregulation of splenic inflammatory cytokines (Ganta et al., 2004), we hypothesized that central ghrelin administration would inhibit splenic SND. Contradictory to the proposed
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Fig. 2. Changes in (A) mean arterial pressure (ΔMAP, mm Hg) and (B) heart rate (ΔHR, bpm) responses to ICV administration of aCSF (n = 13) or ghrelin (1 nmol/kg, n = 12). Time 0 signifies control. ICV ghrelin and aCSF infusions were completed over a 5 min infusion period (shaded in grey). Measurements were maintained for 45 min after administration of ghrelin or aCSF. *Significantly different from control values. †Ghrelin significantly different from aCSF. Values are means ± SE.
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
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S. Balivada et al. / Autonomic Neuroscience: Basic and Clinical xxx (2016) xxx–xxx
The decline in adaptive immune response with aging is largely attributable to the structural and functional involution of the lymphoid organs, which leads to a decline in T-lymphocyte output (Dixit et al., 2009). Both ghrelin and GHS-R1a receptor levels in the thymus demonstrate a progressive decline during aging and this loss of ghrelin activity alters the associated T cell population (Dixit et al., 2009). However administration of ghrelin into old mice rejuvenates the senescent thymus architecture and thymocyte numbers (Vanessa et al., 2013) and reduces proinflammatory cytokines (Dixit et al., 2009). Similarly neuroanatomical studies reveal a progressive, age-associated loss in the sympathetic neural innervation to lymphoid organs (Bellinger et al., 1992). Collectively, these data support the existence of aging-associated changes in sympathoimmune physiological interactions and suggest ghrelin as a pharmacological strategy which may be useful in mitigating agerelated alterations in neural-immune relationships. Anesthesia can modulate SND responses; therefore, the use of an anesthetized preparation in the present study provides an experimental limitation. However, the anesthetic regimen employed in the present experimental protocols is widely used in studies involving recordings of SND, and simultaneous recordings of splenic and lumbar SND are best completed, at least in the hands of the present investigative team, in an anesthetized preparation. Importantly, a fundamental regulatory strategy of the SNS involves the nonuniform regulation of the level of activity in nerves innervating different targets, a potential effect that can only be studied via the completion of multiple nerve recordings. Further understanding of interactions coupling ghrelin, central regulation of the SNS, efferent SND, and ultimately target organ function, provides fundamental information regarding the evolving nature of mechanisms regulating the sympathetic-immune interactions. Disclosures No conflicts of interest are declared by the authors. Acknowledgements Supported by NIH grant AG-041948. References Baatar, D., Patel, K., Taub, D.D., 2011. The effects of ghrelin on inflammation and the immune system. Mol. Cell. Endocrinol. 340, 44–58.
Baron, R., Jänig, W., Kollmann, W., 1988. Sympathetic and afferent somata projecting in hindlimb nerves and the anatomical organization of the lumbar sympathetic nervous system of the rat. J. Comp. Neurol. 275, 460–468. Bellinger, D.L., Ackerman, K.D., Felten, S.Y., Felten, D.L., 1992. A longitudinal study of agerelated loss of noradrenergic nerves and lymphoid cells in the rat spleen. Exp. Neurol. 116, 295–311. Dixit, V.D., Yang, H., Cooper-Jenkins, A., Giri, B.B., Patel, K., Taub, D.D., 2009. Reduction of T cell-derived ghrelin enhances proinflammatory cytokine expression: implications for age-associated increases in inflammation. Blood 113, 5202–5205. Felten, D., Felten, S., Carlson, S., Olschowka, J., Livnat, S., 1985. Noradrenergic and peptidergic innervation of lymphoid tissue. J. Immunol. 135, 755–765. Felten, D.L., Felten, S.Y., Bellinger, D.L., Carlson, S.L., Ackerman, K.D., Madden, K.S., Olschowki, J.A., Livnat, S., 1987. Noradrenergic sympathetic neural interactions with the immune system: structure and function. Immunol. Rev. 100, 225–260. Freeman, J.N., do Carmo, J.M., Adi, A.H., da Silva, A.A. 2013. Chronic central ghrelin infusion reduces blood pressure and heart rate despite increasing appetite and promoting weight gain in normotensive and hypertensive rats. Peptides 42, 35–42. Ganta, C.K., Blecha, F., Ganta, R.R., Helwig, B.G., Parimi, S., Lu, N., Fels, R.J., Musch, T.I., Kenney, M.J., 2004. Hyperthermia-enhanced splenic cytokine gene expression is mediated by the sympathetic nervous system. Physiol. Genomics 19, 175–183. Hosking, K.G., Fels, R.J., Kenney, M.J., 2009. Inhibition of RVLM synaptic activation at peak hyperthermia reduces visceral sympathetic nerve discharge. Auton. Neurosci. 150, 104–110. Kenney, M.J., Ganta, C.K., 2014. Autonomic nervous system and immune system interactions. Commun. Phys. 4, 1177–1200. Kenney, M.J., Ganta, C.K., Fels, R.J., 2013. Disinhibition of RVLM neural circuits and regulation of sympathetic nerve discharge at peak hyperthermia. J. Appl. Physiol. (1985) 115, 1297–1303. Krapalis, A.F., Reiter, J., Machleidt, F., Iwen, K.A., Dodt, C., Lehnert, H., Sayk, F., 2012. Ghrelin modulates baroreflex-regulation of sympathetic vasomotor tone in healthy humans. Am. J. Phys. Regul. Integr. Comp. Phys. 302, R1305–R1312. Lambert, E., Lambert, G., Ika-Sari, C., Dawood, T., Lee, K., Chopra, R., Straznicky, N., Eikelis, N., Drew, S., Tilbrook, A., Dixon, J., Esler, M., Schlaich, M.P., 2011. Ghrelin modulates sympathetic nervous system activity and stress response in lean and overweight men. Hypertension 58, 43–50. Lin, Y., Matsumura, K., Fukuhara, M., Kagiyama, S., Fujii, K., Iida, M., 2004. Ghrelin acts at the nucleus of the solitary tract to decrease arterial pressure in rats. Hypertension 43, 977–982. Matsumura, K., Tsuchihashi, T., Fujii, K., Abe, I., Iida, M., 2002. Central ghrelin modulates sympathetic activity in conscious rabbits. Hypertension 40, 694–699. Prior, L.J., Davern, P.J., Burke, S.L., Lim, K., Armitage, J.A., Head, G.A., 2014. Exposure to a high-fat diet during development alters leptin and ghrelin sensitivity and elevates renal sympathetic nerve activity and arterial pressure in rabbits. Hypertension 63, 338–345. Vanessa, V.d.S., Ana Lúcia, S.R., Thereza, C.D.L., Susana, R.d.B., Rita, R.-V., Rui, D.P., 2013. Ghrelin as a neuroprotective and palliative agent in Alzheimer's and Parkinson's disease. Curr. Pharm. Des. 19, 6773–6790. Wu, R., Dong, W., Qiang, X., Wang, H., Blau, S.A., Ravikumar, T.S., Wang, P., 2009. Orexigenic hormone ghrelin ameliorates gut barrier dysfunction in sepsis in rats. Crit. Care Med. 37, 2421–2426. Yasuda, T., Masaki, T., Kakuma, T., Yoshimatsu, H., 2003. Centrally administered ghrelin suppresses sympathetic nerve activity in brown adipose tissue of rats. Neurosci. Lett. 349, 75–78.
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Short Communication
Effect of ghrelin on regulation of splenic sympathetic nerve discharge Sivasai Balivada a,b,1, Hitesh N. Pawar a,b,⁎,1, Shawnee Montgomery a, Michael.J. Kenney a,b a b
Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, United States Department of Biological Sciences, College of Science, University of Texas at El Paso, El Paso, TX 79968, United States
a r t i c l e
i n f o
Article history: Received 2 May 2016 Received in revised form 22 July 2016 Accepted 18 August 2016 Available online xxxx Keywords: Ghrelin Splenic SND Lumbar SND Intracerebroventricular injection
a b s t r a c t Ghrelin influences immune system function and modulates the sympathetic nervous system; however, the contribution of ghrelin to neural-immune interactions is not well-established because the effect of ghrelin on splenic sympathetic nerve discharge (SND) is not known. This study tested the hypothesis that central ghrelin administration would inhibit splenic SND in anesthetized rats. Rats received intracerebroventricular (ICV) injections of ghrelin (1 nmol/kg) or aCSF. Lumbar SND recordings provided a non-visceral nerve control. The ICV ghrelin administration significantly increased splenic and lumbar SND, whereas mean arterial pressure (MAP) was not altered. These findings provide fundamental information regarding the nature of sympathetic-immune interactions. © 2016 Published by Elsevier B.V.
1. Introduction Ghrelin is a peptide hormone that was originally isolated from the gastrointestinal system and identified as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a) (Baatar et al., 2011). Subsequent investigations have demonstrated that this peptide can influence a number of physiological systems and responses, including the sympathetic nervous system (SNS) (Lambert et al., 2011; Matsumura et al., 2002). Ghrelin receptors are expressed in the central nervous system, especially in brainstem and hypothalamic sites (Wu et al., 2009), and central ghrelin administration in experimental animals modulates sympathetic nerve discharge (SND) regulation. The intracerebroventricular (ICV) administration of ghrelin has been reported to decrease brown adipose tissue SND in rats (Yasuda et al., 2003), reduce renal SND in rabbits (Matsumura et al., 2002), and produce little effect on renal SND in rabbit offspring from mothers who consumed a normal fat diet compared with enhanced ghrelin-induced renal SND responses in rabbit offspring from mothers fed a high fat diet (Prior et al., 2014). Microinjection of ghrelin in the nucleus of the solitary tract reduces renal SND in rats (Lin et al., 2004). In human subjects direct recordings of efferent muscle sympathetic nerve outflow using microneurography have provided information regarding the effect of peripherally-administered ghrelin on SND regulation. Lambert and colleagues reported that intravenous infusion of ghrelin increased muscle
⁎ Corresponding author at: Department of Biological Sciences, College of Science, University of Texas at El Paso, Biosciences Research Building, Room No. 4.216, El Paso, TX 79968, United States. E-mail address: [email protected] (H.N. Pawar). 1 These authors contributed equally to this work.
SND and reduced arterial blood pressure (Lambert et al., 2011). These investigators speculated that combined baroreflex-mediated and central neural effects of ghrelin may play a role in mediating the observed sympathoexcitatory response. Krapalis et al., observed a biphasic muscle SND response to intravenous ghrelin; an initial sympathoexcitation associated with reduced arterial blood pressure, followed by a progressive decline of muscle SND towards control levels, despite a sustained reduction in arterial blood pressure (Krapalis et al., 2012). These findings indicate that peripheral ghrelin administration can modulate the level of muscle SND in human subjects, although the role of central neural ghrelin in mediating these responses remains unclear. A role for ghrelin in modulating immune system function is wellestablished, primarily as an anti-inflammatory agent and an immunoregulatory hormone (Baatar et al., 2011). Numerous bidirectional pathways provide the foundation for communication between the nervous system and the immune system, and the efferent arm of the SNS plays an important role in mediating neural-immune interactions (Kenney and Ganta, 2014). Physiological activation of splenic SND enhances the expression of splenic cytokine and chemokine genes, an effect that is abrogated by splenic nerve denervation (Ganta et al., 2004), indicating that changes in the level of efferent splenic nerve outflow can influence immune function in a peripheral lymphoid organ. It is intriguing to postulate that ghrelin may play a role in neuroimmune interactions by influencing the SNS, however, the effect of ghrelin on the level of splenic SND is not known. Given the role of ghrelin as an anti-inflammatory molecule, and the influence of splenic SND activation to influence splenic cytokine gene expression, the first goal of the present investigation was to test the hypothesis that ICV administration of ghrelin would inhibit splenic SND. Important with respect to the role of ghrelin in modulating muscle SND in human
http://dx.doi.org/10.1016/j.autneu.2016.08.011 1566-0702/© 2016 Published by Elsevier B.V.
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
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subjects (Krapalis et al., 2012; Lambert et al., 2011), to date there is no information available concerning the effect of central ghrelin on the level of lumbar SND, despite the fact that this nerve innervates the rat hindlimb and tail (Baron et al., 1988). In this regard, lumbar SND recordings may provide translational significance to previous studies that tested the effect of ghrelin on muscle SND in human subjects (Krapalis et al., 2012; Lambert et al., 2011). Therefore, the second goal was to determine the effect of ICV administration of ghrelin on the level of lumbar SND. 2. Methods The experimental procedures and protocols were completed in accordance with the American Physiological Society's guiding principles for research involving animals and approved by the Institutional Animal Care and Use Committee at Kansas State University and at the University of Texas at El Paso. 2.1. General procedures Experiments were completed using male Sprague-Dawley rats (419 ± 15 g). Anesthesia was induced by isoflurane (3–5%) and maintained during surgical procedures using isoflurane (1.25%–1.75%), αchloralose (80 mg/kg, iv), and urethane (400 mg/kg, ip) (Kenney et al., 2013). Isoflurane anesthesia was discontinued following completion of the surgical procedures. During experimental protocols, maintenance doses of α-chloralose (35–45 mg/kg/h) were administered intravenously and maintenance doses of urethane (200 mg/kg, every 4 h) were administered intraperitoneally. The adequacy of anesthesia during the initial surgical procedures was indicated by the absence of a withdrawal reflex in response to mechanical stimulation of the tail or hind limb. Femoral arterial pressure was monitored using a pressure transducer connected to a blood pressure analyzer. Colonic temperature was maintained at 37.5–37.8 °C during surgical interventions by a temperature-controlled table. 2.2. Sympathetic nerve recordings Activity was recorded biphasically with a platinum bipolar electrode after preamplification (bandpass filter, 30–3000 Hz) from splenic and lumbar sympathetic nerves. Sympathetic nerves were identified using a lateral approach and dissected free of surrounding connective tissue, and nerve-electrode preparations were covered with silicone gel to prevent exposure to room air. Sympathetic nerve potentials were full-wave rectified, integrated (time constant 10 ms) and quantified as μvolts × seconds (μV·s) (Hosking et al., 2009). SND recordings were corrected for background noise after administration of the ganglionic blocker chlorisondamine (5 mg/kg, iv) or nerve crush. 2.3. ICV injection Anesthetized rats were placed in a stereotaxic frame. The head was leveled between bregma and lambda, a small hole was made in the skull, and the dura was removed to increase precision of the depth of the injector needle. A Hamilton syringe was loaded onto a Quintessential Stereotaxic Injector (Stoelting Co., Wood Dale, IL), and the needle guided to 1.4 mm lateral from midline, 0.9 mm posterior to bregma, and 3.5 mm below dura. The body weight adjusted ghrelin dose was selected based on previous studies that analyzed the effect of ICV ghrelin injection on SND in animal models (Matsumura et al., 2002; Yasuda et al., 2003). Ghrelin (1 nmol/kg; Phoenix Pharmaceuticals) suspended in artificial cerebrospinal fluid (aCSF) or aCSF alone was administered (10 μL) over 5 min. Fluorescent latex microspheres (50 nm diameter, Lumafluor) were administered immediately before euthanasia to histologically verify that injections were completed in the lateral ventricle.
2.4. Experimental protocols After completion of surgical procedures, anesthetized rats were allowed to stabilize for 60 min before initiation of experimental protocols. Splenic SND, lumbar SND, mean arterial pressure (MAP), and heart rate (HR) were recorded continuously throughout experimental protocols. Following stabilization, pre-injection values were averaged over a 15 min control period (time 0). At the end of the control period rats were administered ghrelin (1 nmol/kg) or aCSF via infusions in the lateral ventricle. Infusions were completed over a 5 min period followed by a 45 min post-infusion period. At the end of experiments rats were euthanized by an intravenous overdose of methohexital sodium (Brevital®) (150 mg/kg, iv). 2.5. Data collection and statistical analysis A computer-based ADInstruments Powerlab data acquisition system was used to collect all experimental data. Values are reported as means ± SE. Splenic and lumbar SND data are expressed as percentage change from control values. MAP and HR are reported as the absolute change from control values. Statistical analyses included two-factor (treatment and time) repeated measures ANOVA with one factor repetition (time). Data that demonstrated statistically significant main and interaction effects were further analyzed with Bonferroni t-tests. The overall level of statistical significance was p b 0.05. 3. Results The ICV injection sites were histologically confirmed to be in the lateral ventricle in all experiments included in the present study. Fig. 1 shows summarized splenic SND (A) and lumbar SND (B) data recorded before infusion (time 0), at the completion of the ICV infusion period (5 min), and for 45 min post ICV administration of aCSF (n = 12) or ghrelin (n = 8). Data are presented as change from control levels at 5 min intervals. Splenic and lumbar SND were progressively and significantly increased from control levels after central ghrelin administration (p b 0.05), with the magnitude of the sympathoexcitation more robust in lumbar SND. In contrast, splenic and lumbar SND remained unchanged from control levels for the duration of the post-infusion period following ICV aCSF administration. Fig. 2 shows summarized MAP (A) and HR (B) data recorded before (time 0), at the completion of the ICV infusion period (5 min), and for 45 min after ICV administration of aCSF (n = 13) or ghrelin (n = 12). MAP did not differ from control levels in either aCSF- or ghrelintreated rats, or between groups, during the 45 min post infusion period (Fig. 2A). Similarly, HR did not differ from control levels following aCSF and ghrelin infusions, although in ghrelin-treated rats HR tended to increase whereas in aCSF-treated rats HR tended to decrease. The change in HR between the two treatments became significant at 10 min postinfusion and continued until the end of the post infusion period (p b 0.05) (Fig. 2B). 4. Discussion The present results indicate that ICV ghrelin administration in anesthetized rats produces a progressive excitation of splenic sympathetic nerve outflow, a response pattern paralleled by a robust ghrelininduced lumbar sympathoexcitation. Central ghrelin administration was not associated with significant changes in arterial blood pressure, suggesting that the sympathoexcitatory responses to ICV ghrelin were not secondary to unloading of the arterial baroreceptors. As expected, each of the measured variables remained unchanged from control values in response to the ICV administration of aCSF. These findings support the notion that, under the conditions of the present experiments, administration of ghrelin into the lateral ventricle modulates the central
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
S. Balivada et al. / Autonomic Neuroscience: Basic and Clinical xxx (2016) xxx–xxx aCSF Ghrelin
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Fig. 1. Changes in (A) splenic sympathetic nerve discharge (SND, %Δ) and (B) lumbar SND (%Δ) responses to the ICV administration of aCSF (n = 12) or ghrelin (1 nmol/kg, n = 8). Time 0 signifies control. ICV ghrelin and aCSF infusions were completed over a 5 min infusion period (shaded in grey). Measurements were maintained for 45 min after administration of ghrelin or aCSF. *Significantly different from control values. †Ghrelin significantly different from aCSF. Values are means ± SE.
hypothesis, in the present study ICV ghrelin administration was associated with splenic sympathoexcitation. This finding does not diminish the fact that ghrelin exerts substantial anti-inflammatory physiological responses, which are mediated by a variety of mechanisms including local and systemic effects; rather it indicates that a central ghrelinmediated splenic sympathoinhibition is likely not a key contributor. Based on the ghrelin-induced activation of lumbar and splenic SND, it might have been expected that arterial blood pressure would be increased in response to ICV ghrelin administration. However, this was not the case and one explanation is that central ghrelin administration may produce nonuniform changes in SND, that is; inhibition of renal SND (Matsumura et al., 2002) and activation of lumbar SND and splenic SND (Fig. 1). Directionally opposite changes in the level of activity in nerves innervating vascular beds that receive substantial levels of cardiac output would be expected to moderate changes in mean arterial blood pressure. However, this is not a complete explanation based on the results of other studies. For example, the reduction in renal SND to central ghrelin administration reported by Matsumura et al. (2002) was associated with a concomitant, marked reduction in arterial blood pressure. On the other hand, Freeman et al. (2013) reported that chronic ICV ghrelin infusion in conscious rats caused a modest reduction in arterial blood pressure (SND was not recorded in this study, and ICV ghrelin administration in rabbit offspring from mothers who consumed a normal fat diet produced modest changes in both renal SND and arterial blood pressure (Prior et al., 2014). At the present time, it is difficult to reconcile differences in arterial blood pressure responses to central ghrelin administration between individual studies, although multiple experimental variances, including; species, anesthesia, ghrelin dose, and ghrelin infusion rates, likely contribute.
regulation of SND by increasing the level of activity in sympathetic nerves innervating two distinct targets. The present study is the first to determine the effects of ghrelin on splenic and lumbar SND, thereby extending the current level of knowledge regarding the role of ghrelin as a modulator of sympathetic neural outflow. The sympathetic innervation to the spleen, via the splenic nerve, provides a neural communication pathway from central nervous system to immunocompetent cells in this lymphoid organ (Felten et al., 1985; Felten et al., 1987). Ghrelin-induced activation of lumbar SND provides information regarding the central effect of this peptide hormone on the sympathetic innervation to the rat hind limb, which may be of translational importance as ghrelin administration modulates muscle SND in human subjects (Krapalis et al., 2012; Lambert et al., 2011). The SNS and the immune system are prominent adaptive physiological systems that for many years were considered to function independently of each other. However, recent lines of inquiry have established a role for the SNS in mediating neural-immune interactions (Kenney and Ganta, 2014). Moreover, it is well-established that ghrelin can exert potent anti-inflammatory effects both in vitro and in vivo, with a promising therapeutic outlook in the treatment of inflammatory diseases (Vanessa et al., 2013). A strategic aspect associated with identifying integrative immune-sympathetic physiological interactions is focused on determining if immune system-related molecules engage supraspinal pre-sympathetic motor neurons. Based on the role of ghrelin as an anti-inflammatory molecule, and the capacity of splenic SND activation to mediate an upregulation of splenic inflammatory cytokines (Ganta et al., 2004), we hypothesized that central ghrelin administration would inhibit splenic SND. Contradictory to the proposed
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Fig. 2. Changes in (A) mean arterial pressure (ΔMAP, mm Hg) and (B) heart rate (ΔHR, bpm) responses to ICV administration of aCSF (n = 13) or ghrelin (1 nmol/kg, n = 12). Time 0 signifies control. ICV ghrelin and aCSF infusions were completed over a 5 min infusion period (shaded in grey). Measurements were maintained for 45 min after administration of ghrelin or aCSF. *Significantly different from control values. †Ghrelin significantly different from aCSF. Values are means ± SE.
Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011
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The decline in adaptive immune response with aging is largely attributable to the structural and functional involution of the lymphoid organs, which leads to a decline in T-lymphocyte output (Dixit et al., 2009). Both ghrelin and GHS-R1a receptor levels in the thymus demonstrate a progressive decline during aging and this loss of ghrelin activity alters the associated T cell population (Dixit et al., 2009). However administration of ghrelin into old mice rejuvenates the senescent thymus architecture and thymocyte numbers (Vanessa et al., 2013) and reduces proinflammatory cytokines (Dixit et al., 2009). Similarly neuroanatomical studies reveal a progressive, age-associated loss in the sympathetic neural innervation to lymphoid organs (Bellinger et al., 1992). Collectively, these data support the existence of aging-associated changes in sympathoimmune physiological interactions and suggest ghrelin as a pharmacological strategy which may be useful in mitigating agerelated alterations in neural-immune relationships. Anesthesia can modulate SND responses; therefore, the use of an anesthetized preparation in the present study provides an experimental limitation. However, the anesthetic regimen employed in the present experimental protocols is widely used in studies involving recordings of SND, and simultaneous recordings of splenic and lumbar SND are best completed, at least in the hands of the present investigative team, in an anesthetized preparation. Importantly, a fundamental regulatory strategy of the SNS involves the nonuniform regulation of the level of activity in nerves innervating different targets, a potential effect that can only be studied via the completion of multiple nerve recordings. Further understanding of interactions coupling ghrelin, central regulation of the SNS, efferent SND, and ultimately target organ function, provides fundamental information regarding the evolving nature of mechanisms regulating the sympathetic-immune interactions. Disclosures No conflicts of interest are declared by the authors. Acknowledgements Supported by NIH grant AG-041948. References Baatar, D., Patel, K., Taub, D.D., 2011. The effects of ghrelin on inflammation and the immune system. Mol. Cell. Endocrinol. 340, 44–58.
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Please cite this article as: Balivada, S., et al., Effect of ghrelin on regulation of splenic sympathetic nerve discharge, Auton. Neurosci. (2016), http:// dx.doi.org/10.1016/j.autneu.2016.08.011